Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism

Abstract

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO 2, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe 2+, α-ketoglutarate, O 2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO 2, succinate and Fe 2+, in which Fe 2+ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO 2 is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO 2 and succinate or after the release of all products, including Fe 2+, and dehydroascorbate is released before the binding of Fe 2+. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical. Zn 2+, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe 2+, α-ketoglutarate, O 2 and ascorbate respectively, and epinephrine noncompetitively with regard to all cosubstrates. Apparent K i values are given for the product and other inhibitors.